Development of a stable dual cell-line GFP expression system to study estrogenic endocrine disruptors

Environmental estrogenic endocrine disruptors are a health concern. Here we constructed a dual cell-line green fluorescence protein (GFP) expression system to identify and study endocrine disrupting compounds with activities of estrogen receptor agonists or antagonists. Human breast cancer MCF-7 cells and endometrial carcinoma Ishikawa cells were infected with a two tandem estrogen response elements--E4 promoter-GFP reporter gene construct. The use of GFP reporter enabled direct and simple evaluations of cell responses. GFP intensity in stably transfected MCF7-GFP and Ishikawa-GFP cells was dose-responsive to 17-beta-estradiol, diethylstilbestrol, 2-hydroxyestradiol, and environmental toxins bisphenol A, genistein and o-p'-DDT. Raloxifene and tamoxifen were effective antiestrogens in MCF7-GFP cells, but acted as partial estrogen receptor agonists in Ishikawa-GFP cells at concentrations of 0.1 nM and above. No synergistic effect was observed in chemical combinations between organochlorine pesticides methoxychlor, o-p'-DDT, p-p'-DDT, nor between estradiol and estrone. In summary, for the first time the effects of estrogen receptor agonists or antagonists were compared between mammary and endometrial cancer cells both stably expressing identical plasmids with GFP reporter genes under the control of tandem estrogen response elements. This dual cell-line system provides a rapid method and sensitive assay to identify environmental estrogens, antiestrogens, selective estrogen receptor modulators and to study their tissue specific effects and chemical interactions. Such a system is especially useful for direct and parallel toxicity assessments with a microfluidic cell culture device.     

Regulation of pS2 gene expression by affinity labeling and reversibly binding estrogens and antiestrogens: comparison of effects on the native gene and on pS2-chloramphenicol acetyltransferase fusion genes transfected into MCF-7 human breast cancer cells

We have examined the effects of reversibly and irreversibly binding estrogenic and antiestrogenic ligands for the estrogen receptor on pS2 RNA accumulation in MCF-7 human breast cancer cells and on pS2-chloramphenicol acetyl transferase (CAT) fusion gene expression in transfected MCF-7 cells. In MCF-7 cells grown in the absence of estrogens, the reversibly binding estrogen, estradiol, and the affinity labeling estrogen, ketononestrol aziridine, KNA, evoked a 13-fold increase in pS2 RNA level. The reversibly binding antiestrogen trans-hydroxytamoxifen and the affinity labeling antiestrogens tamoxifen aziridine or desmethylnafoxidine aziridine behaved as partial agonists/antagonists. In thymidine kinase-chloramphenicol acetyltransferase (tk-CAT) fusion genes containing a 1000 base pair fragment of the pS2 5'-flanking region encompassing the estrogen responsive element of the gene [pS2 (-1100/-90) tk-CAT], estradiol and ketononestrol aziridine evoked a marked stimulation of CAT activity and, in transfected cells grown in both the presence or absence of the weak estrogen phenol red, the antiestrogens behaved as partial agonists/antagonists. This pS2 5'-flanking region displayed both estrogen-dependent and estrogen-independent enhancer activity as monitored by stimulation of CAT activity. Hormonal regulation of the transfected pS2 fusion gene was similar to that observed in the native pS2 gene of MCF-7 cells; however, antiestrogens, while still partial agonists-antagonists, were relatively more agonistic on the transfected fusion gene than on the native gene. One antiestrogen (ICI 164,384) that behaved as a pure estrogen antagonist on the native gene was a partial agonist-antagonist of pS2 gene expression in the plasmid. This study illustrates that the hormonal regulation of the pS2 gene, as characterized by the agonist-antagonist balance of estrogens and antiestrogens, is influenced by the DNA context of the pS2 estrogen responsive element. Also, the fact that estrogens and antiestrogens that form covalent bonds with the estrogen receptor modulate activity of the native pS2 gene and the pS2-tk-CAT fusion gene in a manner similar to that of their reversibly binding counterparts suggests that it may be possible to use these irreversibly binding ligands to follow the interaction of hormone-receptor complexes with regions regulating estrogenic stimulation of the pS2 gene.     

Estrogenic properties of isoflavones derived from Millettia griffoniana.

In most developing countries, 70-80% of the population still resort to traditional medicine for their primary health care. This medicine utilises medicinal plants which are traditionally taken as concoction and infusion. The root and stem bark of Millettia griffoniana (Leguminosae), has been reported to contain isoflavonoids, alkaloids, and diterpenoids. The possible benefit of some bioactive isoflavones derived from M. griffoniana prompted us to screen them for estrogenic activity. Six isoflavones and coumarin derived from M. griffoniana (bail) namely, compound nos. 1-6 (Fig. 1) were tested for their potential estrogenic activities in three different estrogen receptor alpha (ERalpha)-dependent assays. In a yeast-based ERalpha assay, all test substances and 17beta-estradiol as endogenous agonist, showed a significant induction of beta-galactosidase activity. The test compounds at the concentration of 5 x 10(-6) M could achieve 59-121% of the beta-galactosidase induction obtained with 10(-8) M 17beta-estradiol (100%). In the reporter gene assay based on stably transfected MCF-7 cells (MVLN cells), the estrogen responsive induction of luciferase was also stimulated by the M. griffoniana isoflavones. In Ishikawa cells, all substances exhibited estrogenic activity revealed by the induction of alkaline phosphatase (AlkP) activity. The estrogenic activities of isoflavones from M. griffoniana could be completely suppressed by the pure estrogen antagonist, ICI 182,780, suggesting that the compounds exert their activities through ERalpha. Although all substances showed estrogenic effects, 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl]isoflavone (7-O-DHF), Griffonianone C (GRIF-C), and 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO) were found to be the most potent of tested substances. In summary, estrogenic activities of the isoflavones derived from M. griffoniana were described for the first time using reporter gene assays and the estrogen-inducible AlkP Ishikawa model.     

Dehydroepiandrosterone stimulates the estrogen response element

Studies suggest that the steroid, dehydroepiandrosterone (DHEA) can exert effects directly, in addition to its indirect role serving as a precursor for other steroids such as androgens and estrogens. Because DHEA is one of the most abundant adrenal steroids secreted in man, we investigated the functional activity of DHEA on the classic estrogen response element (ERE) in the presence of the estrogen receptor (ER) in transiently transfected cells. GT1-7 hypothalamic neuronal cells, devoid of the estrogen receptor, were transiently transfected with the estrogen receptor expression plasmid (HEGO) and the estrogen response element luciferase (ERELUC) reporter vector. As expected, a dose-response stimulation of luciferase activity was observed in cells treated with estradiol. Concentrations of estradiol from 10⁻¹⁰–10⁻⁶ M resulted in a 136–195 percent increase in luciferase activity compared with control. A dose-response stimulation was also observed in the cells treated with DHEA. A maximum stimulation of 177 percent increase in luciferase activity compared with control was observed with DHEA at a concentration of 10⁻⁵ M. Both the estradiol and DHEA stimulation of ERE luciferase activity was inhibited by the estrogen receptor antagonist, ICI 182,780. The aromatase inhibitor, formestane in combination with estradiol or DHEA had no effect on luciferase activity, suggesting that the effect of DHEA is independent of its conversion to estadiol. Estradiol levels, as measured by ELISA, were appropriately elevated in the estradiol-treated cells but were not significantly different from the control cells in the DHEA-treated cells. These studies suggest a functional in vitro role of DHEA in activating the ERE in the presence of the classic ER.     

Stable luciferase transfected cells for studying steroid receptor biological activity

In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in Intact cells using a photon-counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time. Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.     

2,5-Diphenylfuran-based pure antiestrogens with selectivity for the estrogen receptor alpha

The estrogen receptor alpha (ERalpha) is understood to play an important role in the progression of breast cancer. Therefore, pure antiestrogens with a preference for this receptor form are of interest as new agents for the treatment of this malignancy. Several chemical structures with selective binding affinity for ERalpha have been identified and might be useful for the synthesis of ERalpha-selective pure antiestrogens. In this study we applied the 2,5-diphenylfuran system which is closely related to the triphenylfurans described by others. Various side chains with amino and/or sulfur functions were linked to C3 to convert the furans to estrogen antagonists without residual estrogenic activity. The degree of alpha-selectivity which ranges from 2.5- to 236-fold is strongly influenced by the alkyl group at C4. Antiestrogenic potency was determined in MCF-7/2a breast cancer cells stably transfected with a luciferase gene under the control of an ERE. The 2,5-bis(4-hydroxyphenyl)furan with an ethyl substituent and a 6-[N-methyl-N-(3-pentylthiopropyl)amino]hexyl side chain exerted the strongest antiestrogenic effect in this series with an IC(50) value of 50 nM in cells stimulated with 1 nM estradiol. The RBA values of this derivative were 18% (ERalpha) and 3.4% (ERbeta) of estradiol, respectively. It inhibited the growth of wild-type MCF-7 cells with an IC(50) value of 22 nM. The data show that the 2,5-diphenylfuran system is appropriate for the development of pure antiestrogens with preference for ERalpha.     

Differential activation of wild-type estrogen receptor alpha and C-terminal deletion mutants by estrogens, antiestrogens and xenoestrogens in breast cancer cells

17beta-Estradiol (E2), diethylstilbestrol (DES) and several synthetic (or xenoestrogenic) compounds induced transactivation in MCF-7 or MDA-MB-231 cells transfected with wild-type estrogen receptor alpha (ERalpha) and a construct (pERE(3)) containing three tandem estrogen responsive elements (EREs) linked to a luciferase gene. In contrast, the antiestrogens ICI 182,780 and 4-hydroxytamoxifen (4-OHT) were inactive in this assay. We have investigated the effects of these compounds and several structurally-diverse estrogenic compounds on transactivation in cells transfected with pERE(3) and wild-type ERalpha, mutant ERalpha (1-553), and ERalpha (1-537) containing deletions of amino acids 595-554 and 595-538, respectively. These constructs were used to develop an in vitro assay to distinguish between different structural classes of estrogenic compounds. The results obtained using these constructs were highly cell context- and structure-dependent. Neither E2- nor diethylstilbestrol-induced transactivation in MCF-7 (or MDA-MB-231) cells transfected with pERE(3)/ERalpha (1-537) due to partial deletion of helix 12; however, octylphenol and nonlylphenol, resveratrol (a phytoestrogen), kepone and 2',3',4',5'-tetrachloro-4-biphenylol were "estrogenic" in MCF-7 cells transfected with pERE(3)/ERalpha (1-537). Moreover, the structure-dependent estrogenic activities of several synthetic estrogens (xenoestrogens) in MDA-MB-231 cells were different than those observed in MCF-7 cells. These results demonstrate that the estrogenic activity of many synthetic compounds do not require activation function 2 (AF-2) of ERalpha and are mechanistically different from E2. These data suggest that xenoestrogens are selective ER modulators (SERMs).     

Conversion of dehydroepiandrosterone sulfate at physiological plasma concentration into estrogens in MCF-7 cells

Metabolism of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and androstene-3,17-dione (delta(4)) was performed at their physiological plasma concentrations in MCF-7 cell cultures (1 microM, 10 and 2 nM, respectively). Final metabolic products of these steroids were separated by HPLC-radioactive flow detection and identified by LC/MS or MS/MS. Typical and specific mass fragmentation spectra identified the presence of estrone (E(1)), 17beta-estradiol (E(2)), delta(4), DHEA, 5-androstene-3beta,17beta-diol (delta(5)), and testosterone as principal DHEAS metabolites. Other steroids, such as androstenedione, androsterone, and DHEA fatty acid esters at very low concentrations (from pM to nM), were also obtained after steroid incubation. This highly specific method allowed us to conclude whether a metabolite and enzymatic activity of interest were present in MCF-7 cells or not. We also showed that DHEAS at its physiological plasma concentration may be converted into estrogens and estrogen-like compounds in breast cancer cells. The estrogenic action of DHEAS on breast cancer cells was also measured by bioluminescence in a stably transfected human breast cancer MCF-7 cell line with a reporter gene that allowed expression of the firefly luciferase enzyme under the control of an estrogen regulatory element.     

利用Gaussia萤光素酶建立乳腺癌实时定量检测动物模型

目的：建立-种基于分泌型萤光素酶的实时定量检测实验动物体内肿瘤大小的方法。方法：以分泌型Gaussia萤光素酶（Gluc）为报告基因,以嘌呤霉素为筛选基因,将两者用T2A元件连接后克隆到慢病毒载体,包装慢病毒后感染乳腺癌MCF-7细胞,经嘌呤霉素筛选得到稳定转染细胞MCF-7-Gluc,并检测细胞上清中Gluc活性随时问和细胞数目的变化;将MCF-7-Gluc扩大培养后经皮下注射到雌性BALB／c裸鼠前肢腋下,待肿瘤形成后,检测外周血液中Gluc活性与肿瘤体积的相关性。结果：体外实验显示稳定转染细胞MCF-7-Gluc分泌到细胞上清的Gluc活性与时间和细胞数量在-定范围内均呈现良好的线性关系,体内实验显示裸鼠血液中的Gluc活性与肿瘤体积呈正相关。结论：Gluc技术可作为-种灵活、方便、实时定量检测活体动物体内肿瘤大小的有效工具。